The emphasis on phosphorus removal and recovery from wastewater treatment plants has intensified in recent years due to the urgent need to reduce dependency on nonrenewable phosphorus reserves. Enhanced biological phosphorus removal (EBPR), driven by a diverse community of polyphosphate-accumulating organisms with distinct metabolic capabilities, offers several advantages over chemical precipitation methods. These benefits include reduced chemical use, lower sludge volumes, decreased reliance on costly chemical precipitants, and improved phosphorus recovery quality. Recent advancements in recovery technologies now enable efficient phosphorus extraction from digester supernatant, dewatered digested sewage sludge, and sewage sludge ash, each yielding different recovery efficiencies. Despite these advances, a comprehensive assessment of the phosphorus recovery potential from these target streams in conjunction with EBPR remains crucial and has yet to be fully explored.

Optimizing resource use is essential for the survival and fitness of species in microbial communities ubiquitous in natural and engineered ecosystems. These ecosystems are often characterized by the simultaneous presence of multiple substrates such as volatile fatty acids, amino acids and sugars. Yet, the evaluation of metabolic potential for these microbial community members is predominantly based on single substrate utilisation. Metabolic and ecological implications of the interactions of multiple substrates, particularly in environments with changes in redox conditions and substrate availability, remain poorly understood. In this study, we investigate the metabolic interactions resulting from co-substrate utilization in polyphosphate-accumulating organisms within wastewater treatment systems. We combined experimental analysis of highly enriched “Ca. Accumulibacter” mixed cultures with genome-resolved metagenomics and conditional flux balance analysis (cFBA) to quantify the physiological relevance of co-substrate uptake. We observe that anaerobic co-substrate utilisation of acetate and aspartate result in metabolic interactions leading to optimized redox balance, reduced ATP losses and increased biomass yields by up to 8% compared to individual substrate use. Metabolic modelling revealed that these benefits emerge from the network topology, where the interaction of different metabolic routes gives rise to synergistic effects. Extending our analysis to additional substrate pairs, we classify metabolic interactions into three general types: (i) neutral, (ii) one-way synergistic and (iii) reciprocal synergistic. Our findings highlight the importance of metabolic interactions and cellular resource allocation strategies in dynamic microbial ecosystems. This study provides a broader ecological framework for understanding competitive metabolic strategies in environmental organisms. Co-substrate utilization can have direct implications for improving the yield or productivity of bioprocesses.

Extracellular proteins are supposed to play crucial roles in the formation and structure of biofilms and aggregates. However, often little is known about these proteins, in particular for microbial communities. Here, we use two advanced metaproteomic approaches to study the extracellular proteome in a granular Candidatus Accumulibacter enrichment as a proxy for microbial communities that form solid microbial granules, such as those used in biological wastewater treatment. Limited proteolysis of whole granules and metaproteome isolation from the culture's supernatant successfully classified over 50% of the identified protein biomass to be secreted. Moreover, structural and sequence-based classification identified 387 proteins, corresponding to over 50% of the secreted protein biomass, with characteristics that could aid the formation of aggregates, including filamentous, beta-barrel containing, and cell surface proteins. While various of these aggregate-forming proteins originated from Ca. Accumulibacter, some proteins associated with other taxa. This suggests that not only a range of different proteins but also multiple organisms contribute to granular biofilm formation. Therefore, the obtained extracellular metaproteome data from the granular Ca. Accumulibacter enrichment provides a resource for exploring proteins that potentially support the formation and stability of granular biofilms, whereas the demonstrated approaches can be applied to explore biofilms of microbial communities in general.